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Abstract

A vast body of evidence from studies in humans as well as animals illustrates the pivotal role of glucocorticoid signalling during pre- and postnatal lung maturation. Consequently, corticosteroid treatment is the established standard regimen for pre-term infants and has served to reduce incidence and severity of the major complications, respiratory distress syndrome and bronchopulmonary dysplasia. Glucocorticoid effects are mediated by the glucocorticoid receptor (GR) which acts as a ligand-dependent transcription factor and controls target gene expression by DNA-binding-dependent as well as -independent mechanisms. In line with this, disruption of glucocorticoid signalling by germline inactivation of the GR gene in the mouse leads to respiratory failure and postnatal lethality. Intriguingly, mice carrying a point mutation which selectively impairs homodimeric binding of GR to its cognate response elements survive, indicating that the essential functions of GR during murine lung development are mediated via protein-protein interactions rather than DNA-binding. To further elucidate the modes of GR action which mediate these critical effects, conditional gene inactivation was employed taking advantage of the Cre/loxP recombination system. A series of mutant mice was generated, lacking GR in the mesenchyme, endothelial cells or the lung epithelium, respectively, allowing the assessment of the relative contribution of these compartments to the phenotype of the germline mutation. The beneficial effects of corticosteroids have commonly been attributed to their ability to induce the functional maturation of lung epithelial cells including the stimulation of surfactant synthesis as well as sodium and water transport across the epithelium. However, conditional inactivation of the GR gene in all epithelial cells of the developing lung did not impair survival. Although these mutant mice displayed a delayed progression through the late phases of lung maturation, this retardation did not affect respiratory function at birth and was compensated during the first days of life or an artificially prolonged pregnancy. In contrast, mice lacking GR specifically in mesenchymal cells displayed a morphogenetic phenotype strongly reminiscent of GR knockout animals and succumbed to death immediately after birth. Comparable to the germline mutants, lungs of mutant embryos did not proceed through the canalicular and saccular phases of pulmonary development but remained in the pseudoglandular stage until birth. At E18.5, they were characterized by cuboidal epithelial cells and an expansion of the mesenchymal compartment resulting in an almost complete lack of presumptive alveolar airspace. Mutant lungs showed an increased proliferation rate and failed to induce general differentiation markers such as p21cip1. Moreover, the mutation significantly altered the composition of the extracellular matrix which is known to be critical not only as a structural support but also for mesenchymal-epithelial interactions. Finally, endothelium-specific inactivation of the GR gene neither affected postnatal survival nor morphogenetic development of the lung precluding an important function of GR in endothelial cells during the development of the pulmonary vasculature. In summary, the present study demonstrates that GR in the developing murine lung epithelium is not essential for postnatal survival. Instead, critical glucocorticoid effects are mediated by GR action in the mesenchyme which is necessary to promote complete progression through the maturational phases of murine lung development. GR acts particularly in cells of the fibroblast lineage where it controls the composition of the extracellular matrix and is indispensible for the decrease in the general proliferation rate.